The process of gene expression involves two constitutive steps i.e., transcription and translation and leads to the formation of a protein or polypeptide or in some cases RNA with specific functions. The process of transcription is the most important regulatory step in the process of gene expression and its regulation. The initiation of transcription and modulation of gene expression in eukaryotic genes is directed by a variety of DNA sequence elements collectively arranged in a larger sequence called promoter. Promoter is the portion of DNA sequence on 5′ side, i.e. before beginning of the coding region of a gene. It contains the signals for RNA polymerase machinery that initiates transcription and also modulates the level of transcription. Typical eukaryotic promoters consist of two parts—one, called the minimal or core promoter and the other, called upstream regulatory sequences or cis regulatory elements [Odell, J. T., Nagy, F, & Chua N.-H. Nature 313, 810-812 (1985) and Benfey, P. N. & Chua, N.-H. Science 250, 959-966 (1990)]
The minimal promoter or core promoter is a minimal stretch of contiguous DNA sequence that is sufficient to direct accurate initiation of transcription by RNA [Pol II machinery, Smale, S. T, genes dev 15, 2503-2508 (2001)]. A typical core promoter encompasses the site of initiation of transcription with several sequence motifs, which include TATA Box, initiator (Intr) sequence, TFIIB recognition elements (BRE) and other core promoter motifs [Jennifer, E. F. et al., genes & dev 16: 2583-2592 (2002)]. The core promoter provides the site of action to RNA polymerase II which is a multisubunit enzyme with the basal or general transcription factors like, TFIIA, B, D, E, F and H. These factors assemble into a transcription pre initiation complex (PIC) that catalyzes the synthesis of RNA from DNA template.
The activation of the core promoter is done by the additional sequence of regulatory DNA sequence elements to which various proteins bind and subsequently interact with the transcription initiation complex to activate gene expression. These regulatory elements comprise of DNA sequences that individually and/or in combination determine the spatio-temporal expression pattern of a promoter [Benfey, P.N., Ren, L. & Chua, N.-H EMBO 9: 1685-96 (1990)]. These short regulatory elements are located at a varying distance from transcription start point, some regulatory elements (called proximal elements) are adjacent to core promoter while other elements can be positioned several kilobases upstream or downstream of the promoter (enhancers). Both types of promoter elements modulate the level of transcription from core promoter [(Wasylyk B. CRC, Critical Rev. Biochem, 23: 77-120 (1988), Johnson & McKnight Ann. Rev. Biochem. 58: 799-839 (1989), Fussler & Gussin, Method in Enzymology 273: 3-29 (1996)]. Promoters are usually positioned 5′ upstream relative to the transcription start site of coding region of the corresponding gene.
Transgenic plants are developed both, to improve desirable characters (like, yield, disease resistance, phytoremediation etc.) and to use them as protein factories. Often, there is need to introduce multiple genes to achieve the goals of genetic engineering. For example, almost always one gene is used (e.g., resistance to an antibiotics like kanamycin) to allow the selection of transgenic cells and tissues while a second gene is used (e.g., gene for enhancing yield or imparting resistance to a disease etc.) as the target gene to improve quality of the transgenic plant. Each such gene has to be normally expressed from its own promoter sequence. The most widely used constitutive promoters in the development of transgenic plants are the cauliflower mosaic virus 35S transcript promoter, and promoters of the nopaline and octapine synthase, ubiquitin promoter etc. However, introduction of multiple genes of interest into plants using repetitively the same promoter leads to gene silencing. In order to introduce multiple genes through genetic engineering, several strategies have been developed which include, sequential transformation using multiple gene constructs with different selectable markers, co-transformation with multiple constructs, genetic crosses between plants transformed with different constructs etc. One way to reduce the size of plant transformation vector and the number of promoters required while at the same time achieve the expression of multiple genes, will be to develop transcription regulatory signals that can initiate and regulate transcription in both the directions.
The use of bidirectional promoter can certainly overcome the limitation of sequential transformation and co-transformation with two genes. [Xie et al., Nature Biotechnology, 19: 677-679 (2001)] invented a method to convert a naturally occurring unidirectional or polar promoter into a bidirectional promoter. Such promoter can direct the expression of two genes or gene fusions at a time. Only a few types of strong promoters like CaMV 35S promoter have been reported and can be bidirectionalized. There is no earlier report of constructing a completely artificially designed promoter that would not have any long stretch of homology with native genomic DNA. It is very important to develop capability for designing artificial promoters with no sequence homology to genome since such promoters will not be silenced due to homology, [Davies et al., The Plant Journal 12, 791-804 (1997)].
The inventors of this work have earlier established [(U.S. patent application Ser. No. 09/263,692 now U.S. Pat. No. 6,639,065; EP 99301419.0-2106, Sawant et al., 2001, Theor. Appl. Genet. 102, 635-644)] the art of designing artificial synthetic promoters. Such prior synthetic promoter been described in detail in said references and are deemed to be incorporated herein by reference.:
The present invention is partly based on the finding that part of the artificial promoter shown in SEQ ID NO. 1 below:
SEQ ID NO. 1GTCGACCATCATTTGAAAGGGCCTCGGTAATACCATTGTGGAAAAAGTTG GTAATACGGAAAAAGAAGATTCATCATCCAGAAAAGGTGTGGAAAAGTTG TGGATTGCGTGGAAAAAGTTCGATCTGACCATCTCTAGATCGTGGAAAAA GTTCACGTAAGCGCTTACGTACATATGTGGATTGTGGAAAAAGAAGACGG AGGCATCGGTGGAAAAAGAAGCTTGTACGCTGTACGCTGACGATAGATAG ATACACGTGCACGCGTCCACTTGACGCACAATTGACGCACAATGACGCCA CTTGACGCTACTcan be used as an enhancer sequence or a ‘Transcription Activating Module’ (TAM). Another part of the sequence SEQ ID NO. 1, can be used as transcription initiator or a Transcription Initiator Module (TIM). The present invention teachers that the TAM activates and also regulates gene expression in both the directions from the TIM. Artificial synthesis of strong gene expression modules provides a tool to avoid the repetitive use of the so called strong promoters since a large variety of artificial promoters can be designed. Moreover, developing strong bidirectional promoter with multiple tissue specific or inducible characteristics will have major applications in the improvement of desired agronomical traits in plants. Designing of expression vectors for transformation using the so called bidirectional expression module will be of great value for transgenic development and biotechnology industries. The art of designing bidirectional promoters entirely by computational methods, as demonstrated here, provides a great deal of flexibility in genetic engineering.
Construction of synthetic gene expression modules is an important alternative to the dependence on natural promoters. This allows for bypassing gene silencing and also gives capability to regulate and improve expression level of valuable proteins or compounds of particular economic interest in plants. [(Rance, L et al., Plant Science 162: 833-842 (2002), combined three viral promoter sequences to generate highly active promoters that allowed strong transgene expression in plants)].
Authors of the present invention (Sawant S. et al., Theor. Appl. Genet. 102: 635-644) provided the only example for developing a completely artificial synthetic promoter designed for high level of expression in plants. The promoter (U.S. patent application Ser. No. 09/263,692, now U.S. Pat. No. 6,639,065 EP Application No. 99301419.0-2106) was developed by computational methods and demonstrated to express at a high level in a variety of plants and therefore, performed the purpose for which it was designed.
Prior to this invention, there are certain representative patents summarized below, which describe certain bidirectional promoters. U.S. Pat. No. 5,814,618 discloses a bidirectional promoter which has a multiple tet operator sequences. This patent shows that seven repeats of naturally found prokaryotic tet represser/operator/inducer sequences when flanked by two minimal promoters in the presence of tetracyclin inducer could direct the expression of two genes in eukaryotic cells. U.S. Pat. No. 595,564 discloses a bidirectional heterologous but again naturally existing construct for expression of transgenes in plants. U.S. Pat. No. 5,359,142 discloses natural promoter sequences, which have been manipulated to permit variation in enhancement of gene expression. U.S. Pat. No. 5,837,849 discloses yet another natural plant enhancer element that enhances the transcription level of a plant expressible gene. U.S. Pat. No. 5,627,046 discloses a naturally occurring bidirectional promoter. The art of making a eukaryotic polar promoter bidirectional is disclosed in U.S. Pat. No. 6,388,170. The inventors of that patent have used the art to bidirectionlize certain group of naturally occurring plant promoters such as CaMV 35S, PCISV, OPR, and SAG12.